On-chip electrochemical immunoassay platform for specific protein biomarker estimation in undiluted serum using off-surface membrane matrix

The manuscript presents a new biosensor platform using bioreceptors modified porous 2-dimensional (2D) membrane based off-surface matrix for on-chip electrochemical immunoassay. Antibody based bioreceptors modified 2D matrix of porous polycarbonate (PC) membrane with densely packed 20 µm holes as off-surface matrix was incorporated in very close proximity of the sensor surface and integrated with fluidic system for reagent flow and incubation chamber. Covalent attachment of antibodies on 2D PC membrane based off-matrix was achieved using 4-fluoro-3-nitro-azidobenzene (FNAB) cross-linker. Anti-TNF-α/FNAB/PC membrane was integrated over array of micro fingers of gold based sensor chip using double side tape spacer and StartingBlock phosphate buffer saline- Tween-20; (PBS-T20) blocking buffer was utilized to minimize nonspecific binding. Differential pulse voltammetric studies of Anti-Tnf-α/FNAB/PC-Au for protein biomarker (TNF-α) detection and estimation in undiluted serum indicated that the immunosensor system can detect TNF-α linearly in 100 pg/ml to 100 ng/ml range with insignificant interference from other cytokines and serum proteins. Further, immunosensor exhibited high sensitivity of 194 nA/(ng/ml) and 240 nA/(ng/ml), respectively for single and double membrane based system. Thus, use of 2D membrane based off surface matrix may present the new platform to sensitively measure biomarkers electrochemically to pg/ml range with insignificant nonspecific binding and false signal in undiluted serum.